ABSTRACT The flagellated protozoan, Trypanosoma brucei, is a devastating human and veterinary parasite in sub- Saharan Africa, and the causative agent of Human African Trypanosomiasis (HAT). HAT is fatal if untreated, vaccination is not an option, and available drugs are toxic, difficult to administer, and expensive. In the search for new treatments, understanding the basic biology of the parasite is a cornerstone on the path to discovery of unique biological processes that could potentially serve as drug targets. Trypanosomes are exceptional in that they perform gene regulation almost exclusively at posttranscriptional levels, through control of processes such as mRNA stability and translational efficiency. This reliance on posttranscriptional regulation necessitates that RNA binding proteins (RBPs) are the key effectors of trypanosome development, homeostasis, and virulence. Our laboratory discovered DRBD18, an abundant RBP that is essential for the survival of both the human bloodstream form (BF) and the insect vector procyclic form (PF) of T. brucei, and that is not conserved outside the Order Kinetoplastida. DRBD18 depletion in the PF results in significant changes in the abundance of nearly 1000 mRNAs, many of the most highly regulated themselves encoding RBPs and protein kinases. Thus, DRBD18 is positioned at the apex of numerous potential regulatory cascades. Proteomic data suggest that DRBD18 functions in both nuclear mRNA export and translation initiation. Remarkably, the ability of DRBD18 to stabilize or destabilize mRNAs as well as both its protein and mRNA binding specificities are dramatically regulated by arginine methylation, which acts as a molecular switch towards DRBD18 action. In the proposed studies, we will elucidate DRBD18 functions and regulation by 1) identifying direct DRBD18 mRNA targets, 2) mechanistically defining DRBD18 effector pathways, and 3) establishing the methylation-responsiveness of DRBD18 interactions and DRBD18 functions. In Aim 1, we will use iCLIP to define on a genome-wide level the sets of mRNAs that are directly bound by DRBD18 and to determine how these sets of bound mRNAs are regulated upon DRBD18 methylation. In Aim 2, we will determine the roles of DRBD18 in nuclear mRNA export and translation initiation, and test hypotheses regarding regulation of these functions by arginine methylation. In Aim 3, we will study combinatorial RBP interactions and test the hypothesis that methylation-sensitive interactions between DRBD18 and other RBPs regulate the specificity of DRBD18 mRNA targeting. Our studies will define distinct cis-trans modules that mediate methylation-sensitive DRDB18 functions. Using combined genetic, genomic, and biochemical approaches, the proposed studies will provide fundamental insights into specific gene regulatory events in T. brucei, and uncover regulatory mechanisms with wide- ranging applicability in trypanosomes. They also have the potential to broaden our understanding of RNA biology and its regulation by arginine methylation in higher organisms.